In mature human tissues, the ability to initiate angiogenesis (also called “neovascularization”) is typically held under strict control through a balance of pro- and anti-angiogenic factors in the cells. Angiogenesis therefore occurs only under certain controlled circumstances in the adult, such as in wound healing or during certain stages of the menstrual cycle. Uncontrolled or inappropriate angiogenesis in mature organisms can cause a pathogenic condition.
For example, neovascularization of the choroid in the eye causes severe vision loss in patients with age-related macular degeneration (AMD). In diabetic retinopathy (DR), the iris, retina and optic nerve can be damaged by ocular neovascularization. Together, AMD and DR account for the majority of patients suffering from irreversible blindness worldwide. The pathogenic neovascularization seen in both AMD and DR are believed to involve an imbalance between pro- and anti-angiogenic factors in cells of the eye.
Many solid tumors will also initiate angiogenesis to ensure an adequate blood supply. The new blood vessels allow tumors to grow, damaging the surrounding normal tissues. The increased vascularity of the tumors also increases the ability of metastatic tumor cells to colonize distant sites in the body. The angiogenesis initiated by tumors is also thought to involve an alteration of the balance between pro- and anti-angiogenic factors in tumor cells.
Many of the intracellular pro- and anti-angiogenic factors have been identified. The primary pro-angiogenic factor is vascular endothelial growth factor (“VEGF”), also called vascular permeability factor (“VPF”). VEGF exists in at least four different alternative splice forms in humans (VEGF121, VEGF165, VEGF189 and VEGF206), all of which exert similar biological activities. Angiogenesis is initiated when secreted VEGF binds to the Flt-1 and Flk-1/KDR receptors (also called VEGF receptor 1 and VEGF receptor 2), which are expressed on the surface of endothelial cells.
Flt-1 and Flk-1/KDR are transmembrane protein tyrosine kinases, and binding of VEGF initiates a cell signal cascade resulting in the ultimate neovascularization in the surrounding tissue. Flt-1 and Flk-1/KDR are therefore also pro-angiogenic factors.
Another pro-angiogenic factor is the alpha subunit of hypoxia-inducible factor 1 (HIF-1). HIF-1 is a heterodimeric basic-helix-loop-helix-PAS transcription factor consisting of HIF-1 alpha and HIF-1 beta subunits. HIF-1 alpha expression and HIF-1 transcriptional activity increase exponentially as cellular oxygen concentration is decreased.
Yet another pro-angiogenic factor is ICAM-1, which is a 110 kilodalton member of the immunoglobulin superfamily that is typically expressed on a limited number of cells at low levels in the absence of stimulation. Upon stimulation with inflammatory mediators, a variety of cell types (e.g., endothelial, epithelial, fibroblastic and hematopoietic cells) in a variety of tissues express high levels of ICAM-1 on their surface. The interactions of the endothelial cells with the ECM during angiogenesis require alterations of cell-matrix contacts which are caused, in part, by an increase in ICAM-1 expression.
Two further pro-angiogenic factors are angiopoietin-1 (“Ang1”) and angiopoietin-2 (“Ang2”). Ang1 can act in concert with vascular endothelial growth factor (“VEGF”) to promote angiogenesis, although inhibition of Ang1 alone appears to block neovascularization. Ang2 is a context-dependent competitive antagonist of Tie2, but can also activate Tie2 under certain conditions. Thus, Ang2 can be pro- or anti-angiogenic depending on the intracellular environment. The Tie2 receptor can also be considered a pro-angiogenic factor.
Pigment epithelium-derived factor or “PEDF” is a potent anti-angiogenic factor. PEDF was first identified in retinal pigment epithelial cells, but it is also produced by other cells of the eye. Hypoxic conditions in the eye lead to downregulation of PEDF expression, and patients with AMD often lack PEDF in their vitreous.
Another anti-angiogenic factor is angiostatin, which is a proteolytic fragment of plasminogen. Adeno-associated viral vectors expressing angiostatin inhibit angiogenesis in rat and mouse models of ocular neovascularization. Endostatin also has anti-angiogenic properties, as demonstrated by a reduction in the size of laser-induced choroidal neovascularization in mice with high serum levels of endostatin. Subretinal injection of endostatin in a mouse model of retinopathy-of-prematurity also inhibited retinal neovascularization.
A mutant form of the “tissue inhibitor of metalloproteinase-3” or “TIMP-3” gene has been implicated in a macular neovascular disease called Sorsby's fundus dystrophy, and wild-type TIMP-3 has anti-angiogenic properties. Thus, TIMP-3 is considered to be an anti-angiogenic factor. TIMP1, 2 and 4 are also known to be anti-angiogenic factors.
Non-angiogenic diseases or physiological conditions can also result from a change in the relative amounts of certain gene products within a cell. For example, the Bcl-2 gene family includes anti-apoptotic (Bcl-2, Bcl-xL) and pro-apoptotic (Bcl-xS, Bak, Bax) genes. Members of the Bcl-2 family can mediate survival of erythroid cells. Altering the amount of gene products produced from pro- and anti-apoptotic Bcl-2 gene family members can lead to an increase in red cell destruction and anemia. Similarly, if the ratio of Bax to BclxL is increased in a cell, that cell undergoes apoptosis. Induction of apoptosis of specific cell types has implications for directed therapy of diseases such as cancer.
RNA interference (“RNAi”) is a method of post-transcriptional gene regulation that is conserved throughout many eukaryotic organisms. RNAi is induced by small or short (i.e., <30 nucleotide) double stranded RNA (“dsRNA”) molecules which are present in the cell. These short dsRNA molecules, called “small or short interfering RNA” or “siRNA,” cause the destruction of messenger RNAs (“mRNAs”) which share sequence homology with the siRNA. It is believed that the siRNA and the targeted mRNA bind to an RNA-induced silencing complex (“RISC”), which cleaves the targeted mRNA. The siRNA-induced RNAi exhibits multiple-turnover kinetics, with 1 siRNA molecule capable of inducing cleavage of approximately 1000 mRNA molecules. siRNA-mediated RNAi is therefore more effective than currently available technologies for inhibiting expression of a target gene, which bind to the target mRNA or protein in a 1:1 ratio. However, while RNAi can efficiently reduce the amount of cellular factor gene expression in a given cell, it does not increase the amount of anti-angiogenic factors within a cell.
PEDF has been delivered to retinal pigment epithelial cells by adenoviral and adeno-associated viral (AAV) expression vectors, and has reduced the level of experimentally-induced neovascularization in mice. AAV vectors expressing angiostatin and endostatin injected into the eye have also been used to rescue mouse models of ocular neovascularization. Ocular neovascularization in the mouse has also been inhibited by systemically-injected AAV vectors expressing endostatin. The systemically-injected AAV vectors transduce cells of the liver and cause increased serum levels of endostatin. These studies show that anti-angiogenic factors can inhibit ocular neovascularization regardless of whether the factors are produced in the eye or are provided systemically. However, increasing the level of anti-angiogenic factors in a given cell does not remove the pro-angiogenic signals still present within the cells.
What is needed, therefore, are compositions and methods which decrease expression of certain cellular factors and increase the level of other cellular factors in a given cell, in order to control different physiologic states in a subject. Compositions and methods which can both up-regulate anti-angiogenic factors and efficiently down-regulate pro-angiogenic factors in a given cell are particularly desirable.